Disruption of TRF2 association, but not open or closed chromatin, at rDNA in the presence of dnTRF2.

<p>Chromatin from control/uninduced T19 cells and 30 hour dnTRF2-expressing cells was crosslinked with 1% PFA, sonicated to 100–700 bp, and immunoprecipitated with indicated antibodies: (A) H3K4me2, (B) H3K9me3, (C) UBF, and (D) TRF2. Each bar shows relative enrichment as percentage of input by ChIP-PCR. Actin and alpha satellite are control regions. alpha sat  =  alpha satellite (centromere), β-sat  =  distal beta satellite (located telomeric of rDNA), u18S  =  upstream of 18S rDNA region, 18S = 18S rDNA region, IGS  =  intergenic spacer region in rDNA repeat. Error bars show standard error of the mean. (*) indicates significant difference (<i>p</i><0.001) between control and dnTRF2 ChIP enrichment.</p

DNA damage markers appear with increased dnTRF2 expression.

<p>(A) Immunoblot for TRF2, β-actin (loading control), and H2AX-p on whole cell lysates from UV-treated (∼20 J/m²), uninduced/0-hour, 12-hour, 24-hour, 48-hour, and 72-hour dnTRF2-expressing cells. (B) Graphical representation of protein levels measured by arbitrary fluorescence units normalized to β-actin showing increased dnTRF2 protein levels with longer induction periods up to 48 hours. H2AX-p levels increased by 72 hours. (C) Immunoblot for Chk2-p and β-actin showing appearance of phosphorylated Chk2 kinase after 24 hours of dnTRF2 expression.</p

Non-random acrocentric fusion when telomeres are disrupted by various approaches.

<p>(A) Immunoblot of HT1080 whole cell lysates selected for empty vector or shTRF2 retroviral vector for 11 or 15 days with puromycin. Blot shows TRF2 protein as a doublet and β-actin as a loading control. (B) Acrocentric fusions are non-randomly induced in HT1080 cells expressing a retroviral shTRF2 construct for 10 days and 3 weeks. In addition, treatment of cells with the double-strand break inducer zeocin also results in high numbers of acrocentric fusions. asterisk (*) in graph legend denotes observations from dnTRF2 expression previously reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092432#pone.0092432-Stimpson1" target="_blank">[9]</a>.</p

Timing of nucleolar protein disruption with increasing dnTRF2 expression.

<p>(A) Immunostaining of 3D-preserved whole nuclei with nucleolar protein fibrillarin (green) and Ki-67 (red) in control and 45 hour dnTRF2 nuclei show that nucleolar morphologies changes with increased dnTRF2 expression and telomere dysfunction. (B) Induced T19 (dnTRF2-expressing) cells were analyzed at intervals over a 24-hour period using immunofluorescence with antibodies specific to fibrillarin and FLAG (to detect FLAG-tagged dnTRF2 protein - red). Nucleolar changed from a normal spherical shape to less condensed structures resembling nucleolar necklaces. (C) Quantitation of the percent of nuclei showing visibly abnormal nucleolar staining over the timecourse. Abnormal morphology (decondensed, unraveled) of nucleoli (light grey) increased as dnTRF2 was expressed for longer periods. The number of nuclei examined at each timepoint is indicated at the top of each stacked bar. After dnTRF2 expression for 24 hours, there was a statistically significant increase (asterisk) in the proportion of abnormal nucleoli compared to control cells. Scale bars equal 5 micrometers, hr =  hour.</p

Condensin localization decreases on acrocentric short arms in the presence of dnTRF2.

<p>Combined immunostaining-FISH for (A) SMC4 (green) and rDNA (red) on metaphase chromosomes from control and 36 hour dnTRF2-expressing cells. (B) The amount of SMC4 was quantitated by measuring arbitrary fluorescence along the length of the chromosomes and plotting signal intensity as a line plot. A line begins at the p arm (0 on x-axis) and extends to the telomere of the q arm (∼150+ on x-axis). (C) The extent of rDNA and SMC4 co-localization at chromatids of metaphase chromosomes is presented in graphical format. The number of individuals chromatids examined is indicated at the top of each bar. A significant reduction in SMC4 co-localization at rDNA was observed on metaphase chromatids from cells expressing dnTRF2 for 36 hours. Scale bars in (A) are 15 micrometers.</p

dnTRF2 expression correlates with rDNA repeat array dispersion.

<p>The rDNA arrays are located on the short arms of the 5 pairs of acrocentric chromosomes. FISH on RNase-treated nuclei hybridized with an rDNA (green) PAC probe showed that rDNA, normally appearing as multiple punctate foci in the nucleus, becomes more diffuse with increased dnTRF2 expression. The T19 (dnTRF2) cell line contains ∼18 acrocentric chromosomes. Multiple short arms normally converge in the nucleus, so each foci can contain more than rDNA regions from more than one acrocentric chromosome. With increased dnTRF2 expression and telomere dysfunction, the bright foci were reduced, instead appearing as dotted or beaded tracks of fluorescent signals stretching throughout the nucleus. Pseudo-colored and gray-scale single channel images for rDNA are shown below the merged images. Scale bars equal 5 micrometers.</p

H4K20 trimethylation (H4K20me3) at the X centromere in two human cell lines and one of the mouse-human somatic cell hybrids.

<p>Each number across the schematic representation of the centromere is a genomic site that was interrogated by ChIP-PCR. Control regions, including GAPDH, AFM and X-linked ZXDA are also included. The bar graph shows relative enrichment for H3K27 methylation (n = 3 with SD) calculated as percentage of input.</p

H3K9me2 enrichment at X centromeres in human cell lines and mouse-human hybrids.

<p>The schematic shows the structure of the X centromere. Each number along the centromere represents a genomic site or control region that was interrogated by ChIP-PCR with a specific histone antibody. Each bar graph shows relative enrichment for each histone modification (n≥3 with SD). Control regions, including GAPDH, AFM and X-linked ZXDA are also included. The bar graph shows enrichment calculated as percentage of input.</p

CENP-A mapping on X alpha satellite (DXZ1) on normal and partially deleted X chromosomes in a human-hamster somatic cell hybrid.

<p><i>(A)</i> Schematic of the genomic structure of the X centromeres s of the normal X chromosome in the human:hamster cell line HTM18TC8 and of its 2.7 Mb minichromosome derivative retained in the hamster somatic cell hybrid FA3Wg8-4. <i>(B)</i> IF-FISH on two representative chromatin fibers from the normal X chromosome showing that hamster Cenp-A (red) was localized to a portion of DXZ1 (green), and asymmetrically distributed. Each fiber shown represents an independent experiment. Arrowed lines denote Cenp-A staining (red). Scale bar is 10 microns. <i>(C)</i> IF-FISH on two representative chromatin fibers from the truncated X chromosome showed that CENP-A remained asymmetrically distributed on DXZ1. Each fiber shown represents an independent experiment. Scale bar is 5 microns. <i>(D)</i> Genomic sizes of CENP-A binding domains (gray) and DXZ1 arrays (black) on the normal X before and after partial centromere deletion. N = 15 fibers for normal X and n = 20 fibers for deleted X. The asterisk indicates that reduction in CENP-A array size on the deleted X is statisically significant as determined by a Student's t-test (<i>p</i> = 0.01).</p

Primer sets used for ChIP-PCR.

<p>Primer sets used for ChIP-PCR.</p